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A quitosana é um polisacarídeo obtido pela desacetilação das moléculas de quitina, principal constituinte de alguns fungos e do exoesqueleto de crustáceos e insetos. Os grupos amino presentes na quitosana conferem-lhe importantes propriedades biológicas, como a biodegradação, biocompatibilidade, atividade/efeitos imunológicos e atividade antibacteriana. A desacetilação da quitina é um processo cuja conversão é agressiva, já que exige o ataque da quitina em solução de álcalis em alta concentração e à quente, com duração de 6 a 8 horas. Neste trabalho, carapaças de siri da espécie Charybdis hellerii foi fragmentada e pré-tratada para a obtenção da quitosana e cada etapa, desde o pré-tratamento do material in natura à sua conversão em quitosana, foi investigada detalhadamente. Observou-se que dose e taxa de dose não influenciaram no pré-tratamento ou na etapa de desacetilação da quitina; na dose de 20 kGy (gama ou feixe de elétrons), o processo de conversão teve duração de 60 minutos. A quitosana obtida teve baixa massa molar e grau de desacetilação comparável á quitosana padrão (SA), dependendo das condições de irradiação. Além disso, apresentou inibição da atividade bacteriana tanto livre como imobilizada em substratos poliméricos de polipropileno e de polietileno processados também por radiação ionizante. / Chitosan is a polysaccharide obtained from chitins molecule deacetylation, which is the main composition of certain fungi species and crustaceans and insects exoskeleton. The amino groups present in chitosan give it important biological properties such as biodegradability and biocompatibility, activity/immunological effects and antibacterial healing. The deacetylation of chitin is an aggressive process, which reaction processes in 6 to 8 hours under hot concentrated alkali solution. In this work, Charybdis hellerii crab shells was fragmented and pre-treated for chitosan obtention and each conversion step, from in natura material pre-treatment to final chitosan, were investigated in deteails. It was observed dose and dose rate have not influence neither pre-treatment nor chitin deacetylation steps; at 20 kGy (from gamma or electron beam sources), the conversion process was performed in 60 minutes. The obtained chitosan presented low weight and deacetylation degree compared to standard chitosan, considering specific irradiation conditions. Also, obtained chitosan presented bacterial inactivity as a free compoud as immobilized onto polymeric substrates such polypropylene and polyethylene, also processed by ionizing radiation.

A quitosana é um polisacarídeo obtido pela desacetilação das moléculas de quitina, principal constituinte de alguns fungos e do exoesqueleto de crustáceos e insetos. Os grupos amino presentes na quitosana conferem-lhe importantes propriedades biológicas, como a biodegradação, biocompatibilidade, atividade/efeitos imunológicos e atividade antibacteriana. A desacetilação da quitina é um processo cuja conversão é agressiva, já que exige o ataque da quitina em solução de álcalis em alta concentração e à quente, com duração de 6 a 8 horas. Neste trabalho, carapaças de siri da espécie Charybdis hellerii foi fragmentada e pré-tratada para a obtenção da quitosana e cada etapa, desde o pré-tratamento do material in natura à sua conversão em quitosana, foi investigada detalhadamente. Observou-se que dose e taxa de dose não influenciaram no pré-tratamento ou na etapa de desacetilação da quitina; na dose de 20 kGy (gama ou feixe de elétrons), o processo de conversão teve duração de 60 minutos. A quitosana obtida teve baixa massa molar e grau de desacetilação comparável á quitosana padrão (SA), dependendo das condições de irradiação. Além disso, apresentou inibição da atividade bacteriana tanto livre como imobilizada em substratos poliméricos de polipropileno e de polietileno processados também por radiação ionizante. / Chitosan is a polysaccharide obtained from chitins molecule deacetylation, which is the main composition of certain fungi species and crustaceans and insects exoskeleton. The amino groups present in chitosan give it important biological properties such as biodegradability and biocompatibility, activity/immunological effects and antibacterial healing. The deacetylation of chitin is an aggressive process, which reaction processes in 6 to 8 hours under hot concentrated alkali solution. In this work, Charybdis hellerii crab shells was fragmented and pre-treated for chitosan obtention and each conversion step, from in natura material pre-treatment to final chitosan, were investigated in deteails. It was observed dose and dose rate have not influence neither pre-treatment nor chitin deacetylation steps; at 20 kGy (from gamma or electron beam sources), the conversion process was performed in 60 minutes. The obtained chitosan presented low weight and deacetylation degree compared to standard chitosan, considering specific irradiation conditions. Also, obtained chitosan presented bacterial inactivity as a free compoud as immobilized onto polymeric substrates such polypropylene and polyethylene, also processed by ionizing radiation.

Approximately 50 percent of all known peptide hormones are post-translationally modified at their C-terminus. These peptide hormones are responsible for cellular functions critical to survival. Peptidylglycine alpha-amidating monooxygenase (PAM) is a bi-functional enzyme which catalyzes the conversion of peptide pro-hormones to peptide hormones. PAM is the only known mammalian enzyme that catalyzes the necessary alpha-amidation to activate these peptide hormones. PAM has previously been found to perform N-dealkylation, as well as O-dealkylation. We report here that a novel chemistry for PAM, S-dealkylation, has now been shown. PAM was able to catalyzes the hydroxylation and subsequent dealkylation for a series of substituted 2- (phenylthio)acetic acid analogs, leaving a product containing a free thiol capable of coordinating to copper(I). A series of cinnamic acid derivatives have been investigated as turnover dependent inactivators of PAM. It was shown that the inactivating compounds contained electron donating substituents. All compounds bound competitively versus substrate, though no catalytic activity was noted when tested as substrates. Although no Dkinact was observed when using perdeuterated cinnamic acid, one cannot rule out hydrogen abstraction from the Cα as this step may not be rate limiting for inactivation. This suggests that the activated oxygen species generated at CuM may be sufficiently reactive to abstract a hydrogen from an alkene to generate a vinyl radical. Substrate activation is believed to be facilitated by a Cu(II)-superoxo complex formed at CuM. Hydrogen abstraction from the Cα is hypothesized to generate a radical, though this has never been demonstrated spectrometrically. We report here further evidence for the generation of an Ca radical by comparing log(Vmax/KO2) vs σ+ for a series of ring-substituted 4-phenyl-3- butenoic acids. Lastly, a computational study was carried out to probe for a possible binding pocket for the reductant, ascorbate. Though crystal structures have argued that reduction of the enzymebound coppers is collisional, kinetic data for inhibitors competitive against ascorbate indicates that a discrete binding pocket may exist. Our study suggests a specific site for binding and provides free energy calculations in agreement with experimental values for binding constants.

Les C-low threshold mechanoreceptors ou C-LTMRs forment une sous-classe bien particulière de fibres C non-nociceptives innervant exclusivement la peau poilue. En temps normal, leur rôle est de transduire la caresse et détecter le refroidissement. Mais de nouvelles données montrent que les C-LTMRs jouent aussi un rôle important dans la modulation de la douleur, aussi bien inflammatoire que neuropathique. Cependant, les mécanismes moléculaires et cellulaires qui sous-tendent cette dualité sont encore méconnus.Notre équipe a récemment publié des données, issues d'une expérience de séquençage ARN à haut débit, qui précisent l'identité moléculaire des C-LTMRs. A ajouter au trois marqueurs moléculaires déjà connus Tafa4, Tyrosine Hydroxylase et Vglut3, nous avons découvert neuf autres gènes dont l'expression est fortement enrichie dans ces neurones. L'un d'eux est le facteur de transcription bhlha9. Le but de ma thèse étant d'améliorer notre connaissance des C-LTMRs, j'ai notamment étudié le rôle de ce gène dans la somatosensation et la physiologie des C-LTMRs en effectuant un grand nombre de tests comportementaux sur des souris chez qui bhlha9 a été inactivé. De façon intéressante, l'inactivation de bhlha9 entraine un large défaut d'adaptation aux variations de température. De plus, ces souris mutantes présentent une réponse exacerbée à la douleur inflammatoire induite par la formaline. Enfin, cette réponse exacerbée est insensible à l'effet normalement analgésique d'un modulateur positif du système GABAergique ionotropique.En conclusion, bhlha9 est un nouveau marqueur des C-LTMRs nécessaire pour une perception normale de la température et de la douleur inflammatoire. / C-Low Threshold MechanoReceptors (C-LTMRs) are a unique subset of non-nociceptive C-fibers that innervate exclusively hairy skin. At steady-state, these fibers convey low intensity mechanical stimuli (caress and gentle touch) and detect cooling temperatures.Recent data showed C-LTMRs have an important modulatory role in pain processing following tissue injury, both of inflammatory and neuropathic origin. Yet, their molecular and cellular modes of action are for the most part unknown.To provide a better understanding of these C-LTMRs, our team recently published RNA-seq based data that considerably broadens their molecular repertoire. Besides Tyrosine Hydroxylase, V-GluT3 and Tafa4, already known as restricted to C-LTMRs in DRG, we found nine other genes highly enriched in this subset. One of them encodes for the basic Helix-Loop-Helix (bHLH) family transcription factor bHLHa9.To understand the role of bHLHa9 in somatic sensory biology and in C-LTMRs in particular, I have performed a large panel of behavioural tests on mice lacking its expression, from mechanical to thermal stimulation under acute conditions. Interestingly, bHLHa9 knock-out (KO) mice exhibit a broad defect in temperature sensation. Furthermore, these mice present an enhanced inflammatory pain following hindpaw injection of formalin, along with an impaired analgesic response to GABAA receptor positive modulation.In conclusion, bHLHa9 is highly enriched in C-LTMRs and it is required for proper temperature and inflammatory pain perception.

<p> </p><p><strong>Conclusion:</strong>Medical treatment may have a role in platelet count after transfusion. Since the TACSI platelets passed the quality requirements, and the vast majority of patients platelet count increased after TACSI platelet transfusion, the TACSI platelets will replace the old method to produce platelets at the Uppsala University hospital.</p><p> </p><p> </p><p><strong>Methods:</strong> A new approach that pools 8 buffy coats (TACSI platelets) that were separated into 2 units instead of 4-6 buffy coats pooled to 1 unit was investigated in this study. After the platelets were extracted from the buffy coats their quality was controlled and subsequently the platelet product was evaluated in 96 patients.</p><p> </p><p><strong>Results:</strong> The results showed that 80 % of the platelet units passed the European quality requirements. Further, the platelet count was increased in most patients that received TACSI platelets.</p><p><strong> Conclusion:</strong> Medical treatment may have a role in platelet count after transfusion. Since the TACSI platelets passed the quality requirements, and the vast majority of patients platelet count increased after TACSI platelet transfusion, the TACSI platelets will replace the old method to produce platelets at the Uppsala University hospital.</p><p> </p><p> </p><p> </p>

<p>Allelic imbalance, in which two alleles at a given locus exhibit differences in gene expression, chromatin composition and/or protein binding, is a widespread phenomenon in the human and other complex genomes. Most examples concern individual loci located more or less randomly around the genome and thus imply local and gene-specific mechanisms. However, genomic or chromosomal basis for allelic imbalance is supported by multi-locus examples such as those exemplified by domains of imprinted genes, spanning ~1-2 Mb, or by X chromosome inactivation, involving much of an entire chromosome. Recent studies have shown that genes on the two female X chromosomes exhibit a breadth of expression patterns ranging from complete silencing of one allele to fully balanced biallelic expression. Although evidence for heritability of allele-specific chromatin and expression patterns exists at individual loci, it is unknown whether heritability is also reflected in the chromosome-wide patterns of X inactivation.</p><p>The aim of this thesis is to elucidate the extent to which the widespread variable patterns of allelic imbalance on the human X chromosome in females are under genetic control and how access of the transcription machinery to the human inactive X chromosome in females is determined at a genomic level. For the set of variable genes examined in this study, the absence or presence of expression appears to be stochastic with respect to the population rather than abiding by strict genetic rules. Furthermore, variable gene expression that I have detected even among multiple clonal cell lines derived from a single individual suggests fluctuation in transcriptional machinery engagement. I find that, although expression at most genes on the human inactive X chromosome is repressed as a result of X inactivation, a number of loci are accessible to the transcriptional machinery. It appears that RNA Polymerase II is present at alleles on the inactive X even at the promoters of several silenced genes, indicating a potential for expression. </p><p>This thesis embodies a transition in the field of human X chromosome inactivation from gene by gene approaches used in the past to utilizing high-throughput technologies and applying follow-up analytic techniques to draw upon the vast data publicly available from large consortia projects.</p> / Dissertation

En étudiant des modèles de cellules souches, nous avons montré que l'induction des oscillations dans des myocytes ventriculaires normaux inexcitables n'est pas possible en les connectant à des cellules souches. Jusqu'à aujourdhui, cette induction n'a jamais été démontrée même sous de bonnes conditions. Les résultats référencés [5],[4] ne font apparaître qu'une augmentation de la fréquence d'oscillation (soit dans les cellules en culture, soit dans le coeur).<br />Les oscillations ne deviennent possibles que pour les myocytes qui ont un seuil d'excitation des oscillations induites bien plus bas que la normale. Seules les méthodes qui diminuent le niveau d'expression de courant IK1 donnent des résultats. <br />Il existe une autre approche, qui consiste à ne pas connecter directement les cellules souches à des myocytes, mais à d'autres types de cellules cardiaques avec un seuil d'excitation très bas. De cette façon, des oscillations sont induites sans avoir à modifier le courant IK1 . Ces cellules transitoires pourront être des cellules AV node, de Purkinje ou des cellules voisinant SA node.<br />Pour amener un tissus cardiac à oscillation, les pacemakers artificiels de petite taille exigent des courants d'une magnitude bien plus élevée que dans les expériences menées avec des paires de cellules ou des cultures. Pour éviter ce problème, la taille des pacemakers artificiels doit être plus grande que la constante électrotonique ?<br />Pour le courantpacemaker, la plupart des expérimentateurs ont l'habitude de ne mesurer que l'inactivation. Cette seule mesure ne suffit pas pour étudier les oscillations. Les définitions incluant à la fois inactivation et activation du courant pacemaker doivent prévaloir contrairement à la tradition dans le domaine.

Fullerenes have been the focus of significant research effort and curiosity for their unique physicochemical and photochemical properties since their discovery almost 30 years ago. C60 fullerene in particular has received tremendous attention, due to its prevalence in fullerene production and chemical stability. While ambitious prospective applications for C60 have been ubiquitous, the extremely hydrophobic nature of fullerenes and consequent aggregation at the nano scale has hampered many endeavors. Researchers, therefore, have turned their attention to the functionalization of fullerenes to add hydrophilic moieties for applications in aqueous media. It is known that functionalizing the C60 cage alters its innate physicochemical and photochemical properties, but how these changes translate to the properties of C60 aggregates, often termed nC60, is not well understood. Functionalized fullerenes present an intriguing environmental dichotomy. On the one hand C60 has excellent potential as a novel singlet oxygen producing disinfection tool, and on the other the potential toxicological effects of functionalized C60 are largely unknown. With thousands of possible functionalities, a mechanistic understanding of the effects of functionalization is essential. To explore the effects of functionalization on fullerene photochemistry in relevant systems, three types of functional groups were selected and obtained each in series of mono-, bis-, and tris-functionalized forms. Two functionalities contrasted the presence or lack of a quaternary ammonium group and the third was the sterically bulkier phenyl-C61 butyic acid methylester, which is commonly used in polymer photovoltaics. The fullerenes were characterized for innate photochemical properties in organic solvents using UV/Vis, laser flash photolysis, and photochemical degradation experiments. Aqueous aggregates of each derivative were additionally characterized for their physical and chemical properties by dynamic light scattering, transmission electron microscopy, energy dispersive x-ray spectroscopy, and x-ray photoelectron spectroscopy. All derivatives were photoactive when dispersed molecularly in organic solvents, but only the cationic fullerenes showed significant photoactivity as aqueous aggregates. Differences in aggregate size or crystallinity were unable to explain the differential photoactivity between derivatives, contrary to two established hypotheses. Antimicrobial properties were probed using innate toxicity tests and photoinactivation experiments. Again, only the cationic fullerenes were found to exert photochemical action towards Escherichia coli or MS2 bacteriophages. The cationic fullerenes were also innately toxic to E. coli due to the presence of quaternary ammonium moieties. In order to establish a mechanistic understanding of the photochemistry of functionalized C60 aggregates, simulations of the molecular dynamics (MD) were employed and compared with empirical evidences. Simulations provided theoretical values for C60-O2, C60-C60, and C60-H2O interactions for each derivative. Trends observed in the MD results were compared to photochemical characterizations as described above and Raman spectroscopic measurements of C60’s effect on localized water structure. High resolution transmission electron microscopy was used to provide empirical evidence of the C60-C60 interactions. Overall, fullerene aggregate photochemistry is likely driven by aggregate morphology and by intermolecular interactions between fullerenes, water, and O2.

Every waking moment, we make decisions, from where to move our eyes to what to eat for dinner. The ease and speed with which we do this belie the complexity of the underlying neuronal processing. In the visual system, every scene is processed via a complicated network of neurons that extends from the retina through multiple areas in the visual cortex. Each decision requires rapid coordination of signals from the relevant neurons. Deficits in this integration are likely causes of debilitating learning disorders, yet we know little about the processes involved. Previous studies of the macaque visual cortex indicate that as monkeys learn a new task the parts of the brain involved in decision making select which neurons they “listen to”: the most informative neurons become more strongly associated with the animal’s decisions as it learns. However, this process has only been studied over the course of several months as monkeys gradually learn a complex task. We set out to probe the dynamics of this relationship on a shorter timescale. We studied the middle temporal area (MT) of the visual cortex, where neurons are selective for binocular disparity (a depth cue) and motion direction; they have also been shown to contribute to perceptual decisions during motion- and depth-based tasks. After training monkeys on motion and depth detection tasks, we degraded the sensitivity of MT neurons for depth more than motion by reversibly inactivating two major inputs to MT—visual areas V2 and V3—by cooling. We hypothesized that degrading depth information more than motion would lead to bigger changes in the extent to which MT neurons contributed to decisions during the depth task than the motion task. We monitored this contribution to decisions, as measured by detect probability (DP), prior to and during daily inactivation sessions. We found that neuronal DP decreased during the depth task, indicating that neurons became less involved in these decisions. DP did not change during the motion task, suggesting that these changes can be specific to one feature. Our results revealed a level of fast, selective flexibility in the decision circuitry.